Impacts of RNA polymerase II transcription on EJC assembly – EJCbirth

When present downstream a stop codon, the EJC triggers RNA decay. To determine whether excision of specific introns leads or not to EJC deposition, we have cloned several introns downstream the luciferase ORF. If an EJC is loaded, the luciferase mRNA is degraded while in the reverse case the mRNA remains stable. We have constructed several reporters. We are now screening several conditions to modulate transcription (i.e. drugs, mutations). The effects observed will be next confirmed by immunoprecipitations before large-scale analyses.

The study of the luciferase reporters turned out to be more tricky than initially thought. Among 20 introns cloned, only few were kept after notably controlling their splicing in their new sequence environment. Up to know, only the modulation of transcription speed using a polymerase mutant allowed to observe some significant effects. We now have to confirm by immunoprecipitation. To avoid several technical issues due to transfection, we have begun the establishment of stable cell lines with few selected reporters and for the modified polymerases.

The stable cell lines will also serve to project developed in parallel in the group (effect of EJC onto translation). The stable cell lines expressing the polymerase slow or fast will constitute an important milestone to engage large-scale analyses of EJC modulation by RNA pol II transcription.

No production at this stage

The functional outcome of an mRNA is determined by binding proteins acquired at every step along its travel from its transcription sites to the cytoplasm. The impact of nuclear history on mRNA fate is nicely exemplified by the Exon Junction Complex (EJC), a multiprotein complex loaded by the splicing machinery. This splicing tag accompanies mRNAs in the cytoplasm and influences their processing, cellular localization, translation and quality-control by the process of nonsense-mediated mRNA decay (NMD). The EJC is thus a key regulator of mRNA destiny. Recently, we discovered that the EJC is not a constitutive mark of splicing but is differentially loaded on exonic junctions. Preliminary data from large-scale studies by CLIP-seq confirmed that EJC deposition is much more variable than expected in human cells. An important challenge is now to uncover the determinants that regulate EJC deposition.
EJC assembly is tightly linked to the splicing reaction as it starts by the recruitment of EJC core components in active spliceosome, before exon ligation. In vivo, many splicing events are initiated co-transcriptionally, and splicing profiles are regulated by RNA Pol II elongation rates. Indeed, elongation is believed to either affect the recruitment of splicing regulators, or to provide a delay that facilitate splicing at proximal sites. EJCs assembly appears to start co-transcriptionally, and it is thus likely that transcription also contributes to the recruitment of EJC factors. The objective of this original research proposal is to uncover whether modulation of transcription impacts on mRNP assembly and functions via EJC assembly regulation. With novel tools developed by both partners, we will tackle this project from three different and complementary angles.
(i) Does transcription affect EJC assembly on reporter genes? We will design a series of luciferase reporters carrying in their 3' UTR different intron-containing sequences (ICS) selected on the basis of the CLIP-seq data. If the ICS leads to EJC assembly, the mRNA will be degraded by NMD. So, EJC assembly will be monitored by measuring luciferase activity and then controlled by measuring mRNA abundance and performing co-immunoprecipitations. The reporters will be stably expressed in HeLa cells under the control of 5 different promoters known to differently affect splicing. In addition, transfected cells will be treated with drugs that modulate RNA pol II elongation rate and processivity (i.e. DRB, camptothecin).
(ii) Genome-wide analysis of how transcription affects EJC deposition. In parallel, we will globally affect RNA pol II activity with drugs or UV irradiation and establish the transcriptomic map of EJC binding sites under these conditions by CLIP-seq. Hence, we will obtain an extended view of how transcription affects EJC assembly and consequently gene expression, for instance through the modulation of mRNA stability. Based on this new map, we will use a siRNA-mediated transcriptional gene silencing to specifically modify the chromatin structure of selected genes and determine how epigenetic marks influence transcription and EJC assembly.
(iii) Determination of transcription kinetics parameters in relation with EJC assembly. Measuring transcription kinetics in vivo has been very difficult. We and others have developed tools that provide a quantitative measure of initiation frequencies, elongation rates and pauses. These methods use single molecules approaches in fixed and live cells, using highly efficient in situ hybridization and novel fluorescent RNA tags, and they can provide a direct measure of Pol II activity on well-defined, single copy reporter genes. We will thus obtain a precise picture of transcription for reporters that do, or do not, assemble EJCs, thereby providing a better understanding of the mechanisms leading to its assembly.